PROJECT SUMMARY Extraocular muscles (EOMs) are a group of highly specialized skeletal muscles that control eye movements. While body and limb muscles develop from the somites, the EOMs derive from the head mesoderm. EOMs possess a unique quality of being spared in Duchenne muscular dystrophy and in dystrophin-null animal models, contrary to the severe early- or late-onset damage to most other muscles. Distinctively, the ocular accessory muscles (levator palpebrae superioris and retractor bulbi) that are of the same head mesoderm origin as the EOMs, do exhibit dystrophin-null pathology. Although it has been the focus of many investigations, the mechanism behind this EOM sparing has remained unresolved. New insights into unique features of the EOMs can reveal inherent strategies involved in the selective protection of this muscle group and contribute to our knowledge of both pathogenesis and treatment of muscular dystrophy. The proposed project is driven by our novel discovery of a secondary myogenic lineage that specifies the EOM orbital layer formed following birth. The characteristic compartmentalization of the EOMs into orbital and global layers is critical for fine-tuning of eye movements. An analysis of a transgenic Myh11-Cre driver mouse (Myh11 = myosin heavy chain 11, considered a specific marker of terminally differentiated smooth muscle cells) crossed with the R26mTmG (Cre-driven) reporter, has led us to the unexpected detection of a secondary population of myofibers and satellite cells (Pax7+) in the EOMs that emerge following birth and persist to the adult stage. While in wildtype EOMs, this newly detected myogenic lineage occupies primarily the orbital layer, our pilot study suggests that in dystrophin-null mice this lineage is spreading into the global layer, and thus, could be involved in EOM sparing. Gaining insight into this novel secondary myogenic lineage is also significant for a better understanding of EOM functional histogenesis. The immediate aim of this R21 application is to establish and evaluate several Cre/loxP mouse models for the impact of Myh11-Cre driven genetic ablation of the myogenic-relevant transcription factors ? Pitx2 and Pax7 ? on the emergence of the EOM secondary myogenic lineage and the development of the orbital layer. Embryological studies have determined that Pitx2 is mandatory for the establishment of the EOMs while Pax7 is not, but their role in the neonatal orbital layer specification has yet to be addressed. Subsequently, the optimal Cre/loxP model for interfering with the development / function of the secondary myogenic lineage will be crossed onto mdx strain to study the impact on the EOM dystrophin-null phenotype. In all, the proposed study has the potential to provide a foundation for larger scale investigations that would refine our understanding of mechanisms involved in eye muscle functional specification and the sparing of the EOMs in muscular dystrophy, which in turn can provide significant insight toward strategies for ameliorating muscular dystrophy.